Microbial Profiling

Quick Glimpse

  • We use snakemake workflow engine1 to maintain reproducibility which is important in technical validation, regeneration of results and improvement of the microbiome bioinformatics analysis.

Primary references

  1. Mothur 16S v4 Analysis Pipeline2.
  2. Mothur-based SILVA reference files3.
  3. Mothur-based RDP reference files4. Note: The RDP database is to classify 16S rRNA gene sequences to the genus level.
  4. ZymoBIOMICS Microbial Community Standard (Cat # D6306)5. The ZymoBIOMICS Microbial Community DNA Standard is designed to assess bias, errors and other artifacts after the step of nucleic acid purification.

Classification options

Operational Taxonomic Units (OTUs)

  • OTUs are clusters with sequence similarity and are accepted as an analytical unit used in microbial profiling using 16S rRNA gene markers.

Phylotype

  • A phylotype in microbiome research is a DNA sequence or group of sequences sharing more than an arbitrarily chosen level of similarity of a 16S rRNA gene marker.

Amplicon Sequence Variant (ASV)

  • An ASV in microbiome research is any one of the inferred single DNA sequences recovered from a bioinformatics analysis of 16S rRNA marker genes.

Microbial Phylogenies

  • Microbial phylogenies are estimated from gene sequence homologies and the most-likely evolutionary histories are commonly inferred using models of mutation.




Improved Mothur Snakemake Workflow



Example of common FAQs (in progress)

  1. Mothur dist.seqs taking too long.
    • Length of the merged reads (merged reads over 300pb).
    • Not enough overlaping when merging the paired reads.
    • Use other pre-cluster algorithm like Deblur.
    • Too many uniques?
    • Not enough computer power?: Use HPC or Cluster.
    • Try to make contigs using maxee instead of deltaq or use phylogeny approach for downstream analysis, or use clister.split based on taxonomy.
    • do not forget to use controls.



References

1. Köster, J., Mölder, F., Jablonski, K. P., Letcher, B., Hall, M. B., Tomkins-Tinch, C. H., Sochat, V., Forster, J., Lee, S., Twardziok, S. O., Kanitz, A., Wilm, A., Holtgrewe, M., Rahmann, S., & Nahnsen, S. (2021). Sustainable data analysis with snakemake. F1000Research, 10. https://doi.org/10.12688/f1000research.29032.2
2. Close, W. L. (2020). Mothur 16S v4 analysis pipeline. https://github.com/wclose/mothurPipeline
3. Mothur-based silva reference files. https://mothur.org/wiki/silva_reference_files/
4. Mothur-based RDP reference files. https://mothur.org/wiki/rdp_reference_files/
5. ZymoBIOMICS microbial community DNA standard (cat # D6306). https://www.zymoresearch.com/zymobiomics-community-standard